Pseudomonas boanensis sp. nov., a bacterium isolated from river water used for household purposes in Boane District, Mozambique.

A Gram-negative rod with a single polar flagellum was isolated from a freshwater reservoir used for household purposes in Boane District, near Maputo, Mozambique, and designated as strain DB1T. Growth was observed at 30-42 °C (optimum, 30-37 °C) and with 0.5-1.5 % NaCl. Whole-genome-, rpoD- and 16S rRNA-based phylogenies revealed this isolate to be distant from other Pseudomonas species with Pseudomonas resinovorans, Pseudomonas furukawaii and Pseudomonas lalkuanensis being the closest relatives. Phenotypic analyses of strain DB1T showed marked differences with respect to type strains P. resinovorans CCUG 2473T, P. lalkuanensis CCUG 73691T, P. furukawaii CCUG 75672T and Pseudomonas otiditis CCUG 55592T. Taken together, our results indicate that strain DB1T is a representative of a novel species within the genus Pseudomonas for which the name Pseudomonas boanensis is proposed. The type strain is DB1T (=CCUG 62977T=CECT 30359T).

Acinetobacter portensis sp. nov. and Acinetobacter guerrae sp. nov., isolated from raw meat.

The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Portugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acinetobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA-DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T).

Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

"Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n = 9), the Czech Republic (n = 2), and Germany (n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

Acquired genetic mechanisms of a multiresistant bacterium isolated from a treatment plant receiving wastewater from antibiotic production.

The external environment, particularly wastewater treatment plants (WWTPs), where environmental bacteria meet human commensals and pathogens in large numbers, has been highlighted as a potential breeding ground for antibiotic resistance. We have isolated the extensively drug-resistant Ochrobactrum intermedium CCUG 57381 from an Indian WWTP receiving industrial wastewater from pharmaceutical production contaminated with high levels of quinolones. Antibiotic susceptibility testing against 47 antibiotics showed that the strain was 4 to >500 times more resistant to sulfonamides, quinolones, tetracyclines, macrolides, and the aminoglycoside streptomycin than the type strain O. intermedium LMG 3301T. Whole-genome sequencing identified mutations in the Indian strain causing amino acid substitutions in the target enzymes of quinolones. We also characterized three acquired regions containing resistance genes to sulfonamides (sul1), tetracyclines [tet(G) and tetR], and chloramphenicol/florfenicol (floR). Furthermore, the Indian strain harbored acquired mechanisms for horizontal gene transfer, including a type I mating pair-forming system (MPFI), a MOBP relaxase, and insertion sequence transposons. Our results highlight that WWTPs serving antibiotic manufacturing may provide nearly ideal conditions for the recruitment of resistance genes into human commensal and pathogenic bacteria.

Prevotella jejuni sp. nov., isolated from the small intestine of a child with coeliac disease.

Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 : 27, CD3 : 28(T), CD3 : 33, CD3 : 32 and CD3 : 34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 : 28(T) and CD3 : 33, between CD3 : 32 and Prevotella histicola CCUG 55407(T), and between CD3 : 34 and Prevotella melaninogenica CCUG 4944B(T). Strains CD3 : 27, CD3 : 28(T) and CD3 : 33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase ß-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 : 27, CD3 : 28(T) and CD3 : 33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28(T) ( = CCUG 60371(T) = DSM 26989(T)) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 °C.

Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tap.

Acinetobacter spp. are ubiquitous bacteria in the environment. Acinetobacter spp. isolated from a municipal drinking water treatment plant and from connected tap water were identified to the species level on the basis of rpoB gene partial sequence analysis. Intraspecies variation was assessed based on the analysis of partial sequences of housekeeping genes (rpoB, gyrB, and recA). Antibiotic resistance was characterized using the disk diffusion method and isolates were classified as wild or non-wild type (non-WT), according to the observed phenotype. The strains of Acinetobacter spp. were related to 11 different validly published species, although three groups of isolates, presenting low rpoB sequence similarities with previously described species, may represent new species. Most of the isolates were related to the species A. johnsonii and A. lwoffii. These two groups, as well as others related to the species A. parvus and A. tjernbergiae, were detected in the water treatment plant and in tap water. Other strains, related to the species A. pittii and A. beijerinckii, were isolated only from tap water. Most of the isolates (80 %) demonstrated wild type (WT) to all of the 12 antibiotics tested. Non-WT for tetracycline, meropenem, and ceftazidime, among others, were observed in water treatment plant or in tap water samples. Although, in general, this study suggests a low prevalence of acquired antibiotic resistance in water Acinetobacter spp., the potential of some species to acquire and disseminate resistance via drinking water is suggested.

Strain-level typing and identification of bacteria using mass spectrometry-based proteomics.

Because of the alarming expansion in the diversity and occurrence of bacteria displaying virulence and resistance to antimicrobial agents, it is increasingly important to be able to detect these microorganisms and to differentiate and identify closely related species, as well as different strains of a given species. In this study, a mass spectrometry proteomics approach is applied, exploiting lipid-based protein immobilization (LPI), wherein intact bacterial cells are bound, via membrane-gold interactions, within a FlowCell. The bound cells are subjected to enzymatic digestion for the generation of peptides, which are subsequently identified, using LC-MS. Following database matching, strain-specific peptides are used for subspecies-level discrimination. The method is shown to enable a reliable typing and identification of closely related strains of the same bacterial species, herein illustrated for Helicobacter pylori .

Lachnoanaerobaculum gen. nov., a new genus in the Lachnospiraceae: characterization of Lachnoanaerobaculum umeaense gen. nov., sp. nov., isolated from the human small intestine, and Lachnoanaerobaculum orale sp. nov., isolated from saliva, and reclassification of Eubacterium saburreum (Prevot 1966) Holdeman and Moore 1970 as Lachnoanaerobaculum saburreum comb. nov.

Two novel obligately anaerobic, Gram-stain-positive, saccharolytic and non-proteolytic spore-forming bacilli (strains CD3:22(T) and N1(T)) are described. Strain CD3:22(T) was isolated from a biopsy of the small intestine of a child with coeliac disease, and strain N1(T) from the saliva of a healthy young man. The cells of both strains were observed to be filamentous, approximately 5 to >20 µm long, some of them curving and with swellings. The novel organisms produced H(2)S, NH(3), butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98% sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum and four other Lachnospiraceae bacterium-/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium species are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089(T) confirmed that the bacterium is indeed able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, strains CD3:22(T) and N1(T) represent novel species of a new and distinct genus, named Lachnoanaerobaculum gen. nov., in the family Lachnospiraceae [within the order Clostridiales, class Clostridia, phylum Firmicutes]. Strain CD3:22(T) (=CCUG 58757(T) =DSM 23576(T)) is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1(T) (=CCUG 60305(T)=DSM 24553(T)) is the type strain of Lachnoanaerobaculum orale sp. nov. Moreover, Eubacterium saburreum is reclassified as Lachnoanaerobaculum saburreum comb. nov. (type strain CCUG 28089(T) =ATCC 33271(T) =CIP 105341(T) =DSM 3986(T) =JCM 11021(T) =VPI 11763(T)).

Corynebacterium sanguinis sp. nov., a clinical and environmental associated corynebacterium.

Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO2. They contain mycolic acids, major respiratory menaquinones, MK-8 (II-H2) and MK-9 (II-H2), and polar lipids, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, glycolipids and a novel lipid that remains to be characterized and identified. Strains of Corynebacterium sanguinis are genotypically most similar to Corynebacterium lipophiliflavum, with 16S rRNA gene sequence similarities of 98.3% and rpoB sequence similarities of 94.9-95.2%. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were able to clearly differentiate Corynebacterium sanguinis from the most closely related species. The genome size of Corynebacterium sanguinis is 2.28-2.37Mbp with 65.1-65.5mol% G+C content. A total of 2202-2318 ORFs were predicted, comprising 2141-2251 protein-encoding genes. The type strain is CCUG 58655T (=CCM 8873T=NCTC 14287T).

Scandinavium goeteborgense gen. nov., sp. nov., a New Member of the Family Enterobacteriaceae Isolated From a Wound Infection, Carries a Novel Quinolone Resistance Gene Variant.

The family Enterobacteriaceae is a taxonomically diverse and widely distributed family containing many human commensal and pathogenic species that are known to carry transferable antibiotic resistance determinants. Characterization of novel taxa within this family is of great importance in order to understand the associated health risk and provide better treatment options. The aim of the present study was to characterize a Gram-negative bacterial strain (CCUG 66741) belonging to the family Enterobacteriaceae, isolated from a wound infection of an adult patient, in Sweden. Initial phenotypic and genotypic analyses identified the strain as a member of the family Enterobacteriaceae but could not assign it to any previously described species. The complete 16S rRNA gene sequence showed highest similarity (98.8%) to four species. Whole genome sequencing followed by in silico DNA-DNA similarity analysis and average nucleotide identity (ANI) analysis confirmed that strain CCUG 66741 represents a novel taxon. Sequence comparisons of six house-keeping genes (16S rRNA, atpD, dnaJ, gyrB, infB, rpoB) with those of the type strains of the type species of related genera within the family Enterobacteriaceae indicated that the strain embodies a novel species within the family. Phylogenomic analyses (ANI-based and core genome-based phylogeny) showed that strain CCUG 66741 forms a distinct clade, representing a novel species of a distinct, new genus within the family Enterobacteriaceae, for which the name Scandinavium goeteborgense gen. nov., sp. nov. is proposed, with CCUG 66741T as the type strain (= CECT 9823T = NCTC 14286T). S. goeteborgense CCUG 66741T carries a novel variant of a chromosomally-encoded quinolone resistance gene (proposed qnrB96). When expressed in Escherichia coli, the qnrB96 gene conferred five-fold increase in minimum inhibitory concentration against ciprofloxacin. This study highlights the importance and the utility of whole genome sequencing for pathogen identification in clinical settings.

Methicillin-resistant Staphylococcus argenteus misidentified as methicillin-resistant Staphylococcus aureus emerging in western Sweden.

Two strains included in a whole-genome sequencing project for methicillin-resistant Staphylococcus aureus (MRSA) were identified as non-Staphylococcus aureus when the sequences were analysed using the bioinformatics software ALEX (www.1928diagnostics.com, Gothenburg, Sweden). Sequencing of the sodA gene of these strains identified them as Staphylococcus argenteus. The collection of MRSA in western Sweden was checked for additional strains of this species. A total of 18 strains of S. argenteus isolated between 2011 and December 2017 were identified.

Proteotyping: Proteomic characterization, classification and identification of microorganisms--A prospectus.

Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels.

Genotypic and phenotypic applications for the differentiation and species-level identification of achromobacter for clinical diagnoses.

The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected 'house-keeping' genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data.

Achromobacter animicus sp. nov., Achromobacter mucicolens sp. nov., Achromobacter pulmonis sp. nov. and Achromobacter spiritinus sp. nov., from human clinical samples.

The phenotypic and genotypic characteristics of fourteen human clinical Achromobacter strains representing four genogroups which were delineated by sequence analysis of nusA, eno, rpoB, gltB, lepA, nuoL and nrdA loci, demonstrated that they represent four novel Achromobacter species. The present study also characterized and provided two additional reference strains for Achromobacter ruhlandii and Achromobacter marplatensis, species for which, thus far, only single strains are publicly available, and further validated the use of 2.1% concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequence divergence as a threshold value for species delineation in this genus. Finally, although most Achromobacter species can be distinguished by biochemical characteristics, the present study also highlighted considerable phenotypic intraspecies variability and demonstrated that the type strains may be phenotypically poor representatives of the species. We propose to classify the fourteen human clinical strains as Achromobacter mucicolens sp. nov. (with strain LMG 26685(T) [=CCUG 61961(T)] as the type strain), Achromobacter animicus sp. nov. (with strain LMG 26690(T) [=CCUG 61966(T)] as the type strain), Achromobacter spiritinus sp. nov. (with strain LMG 26692(T) [=CCUG 61968(T)] as the type strain), and Achromobacter pulmonis sp. nov. (with strain LMG 26696(T) [=CCUG 61972(T)] as the type strain).

Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively.

The phenotypic and genotypic characteristics of seventeen Achromobacter strains representing MLST genogroups 2, 5, 7 and 14 were examined. Although genogroup 2 and 14 strains shared a DNA-DNA hybridization level of about 70%, the type strains of both genogroups differed in numerous biochemical characteristics and all genogroup 2 and 14 strains could by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence which identified genogroups 2 and 14 as clearly distinct populations, the availability of nrdA sequence analysis as a single locus identification tool for all Achromobacter species and genogroups, and the differential phenotypic characteristics, we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845(T) [=CCUG 62426(T)] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852(T) [=CCUG 62438(T)] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857(T) [=CCUG 62444(T)] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840(T) [=CCUG 62421(T)] as the type strain).

Stenotrophomonas interspecies differentiation and identification by gyrB sequence analysis.

Stenotrophomonas species are found commonly in environmental and clinical samples; Stenotrophomonas maltophilia is an important opportunistic pathogen of humans. Traditional phenotyping protocols, as well as genotyping by 16S rRNA gene sequence analysis, do not reliably distinguish the species of Stenotrophomonas. Sequence analyses of two targeted PCR-amplified regions of the gyrB gene, which encodes the ß-subunit of DNA gyrase, enabled resolution and identification of these species. Most type strains of the different species of Stenotrophomonas exhibited more than 7% dissimilarity in the gyrB gene sequences. Among these, strains identified as the same species exhibited sequence dissimilarities up to 4.6% and 5.9% for the two regions, respectively. Strains identified as S. maltophilia, with 16S rRNA gene sequence similarities > 99.0%, were grouped within a 'S. maltophilia complex'; these organisms exhibited gyrB similarities as low as 93%. Many of these strains possessed genomic DNA similarities with the type strain of S. maltophilia CCUG 5866(T) below 70%. These data, including gyrB sequence comparisons, indicate that strains identified as S. maltophilia may comprise distinct, new species.